The importance of long chain polyunsaturated fatty acids (PUFAs) is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin Nutr., 28:958-966 (1975); Dyerberg, J. et al., Lancet, 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001)). And, numerous other studies document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
A variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high level production of e.g., arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) PUFAs. Whether ω-3/ω-6 PUFA production is the result of natural abilities or recombinant technology, both strategies may require conversion of oleic acid (18:1) to LA by the action of a Δ12 desaturase; ω-3 PUFA production is typically enhanced by the conversion of LA to ALA by the action of a Δ15 desaturase. Subsequent longer-chain PUFAs are generally synthesized via either the Δ6 desaturase/Δ6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by production of γ-linoleic acid (GLA; 18:3 ω-6) and/or stearidonic acid (STA; 18:4 ω-3)) or the Δ9 elongase/Δ8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by production of eicosadienoic acid (EDA; 20:2 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3)) (FIG. 1).
Based on the role that Δ12 desaturase and Δ15 desaturase enzymes play to thereby effectively “push” carbon into the ω-3/ω-6 PUFA biosynthetic pathway, there has been considerable effort to identify and characterize these enzymes from various sources. Although a variety of fungal Δ12 desaturases have been publically disclosed, a limited number of fungal Δ15 desaturases with an unexpectedly high degree of sequence homology to fungal Δ12 desaturases have been described only recently. More specifically, many fungal Δ15 desaturases were initially described as a “Δ12 desaturase-like” protein or polypeptide, based on their significant similarity with known fungal Δ12 desaturases (PCT Publications No. WO 2005/047485 and No. WO 2005/047480).
PCT Publication No. WO 2003/099216 (Monsanto Technology, LLC) teaches fungal sequences and their expression, and specifically includes data supporting the functional characterization of desaturases having Δ15 activity from Neurospora crassa and Aspergillus nidulans, as well as some amino acid motifs derived thereof; a putative “Δ15 desaturase” sequence from Botrytis cinerea is also disclosed. PCT Publication No. WO 2006/019192 describes the Δ15 desaturase of Mortierella alpina. Additionally, Kainou et al. (Yeast, 23(8):605-612 (2006)) and Murayama et al. (Microbiol., 152(5):1551-1558 (2006)) independently characterized Δ12 and Δ15 desaturases from Kluyveromyces lactis and Candida albicans, respectively. Kainou et al. suggests amino acid alterations responsible for the substrate preferences between the Kluyveromyces lactis Δ12 and Δ15 desaturase.
Relatively few fungal Δ15 desaturases are known. Additionally, no facile sequence-based method is available to facilitate the distinction between Δ15 and Δ12 desaturase sequences. The problem to be solved, therefore, is to provide a sequence-based method that easily distinguishes polypeptides having Δ15 desaturase activity as opposed to Δ12 desaturase activity. Applicants have solved the stated problem via a sequence of empirical steps comprising: (1) isolating a pool of Δ12/Δ15 desaturase-like polypeptides of fungal origin; (2) developing a sequence-based means to distinguish fungal Δ12 desaturases from fungal Δ15 desaturases; and, (3) identifying a specific amino acid residue(s) that enables one to alter fungal desaturase enzyme activity, substrate specificity and Δ12/Δ15 regiospecificity.